Anemia Lab

Polychromasia:  variation in staining, and it is likely that these polychromatic, large cells are reticulocytes

Anisocytosis:  variation in size of RBCs and is commonly found as a result of a regenerative response.  This is typically due to the presence of large, immature RBCs.

If this slide had been stained with NMB instead of DQ, the large basophilic red cell at the end of the pointer would likely contain organelles designating it as a reticulocyte.  

"Macrocytes" also sometimes called polychromatophils are usually young, polychromatophilic erythroctyes and may be increased during a responsive anemia

Oxidation of RBCs may occur during some disease states and as well as exposure to some drugs or toxins.  This oxidation an denaturation of hemoglobin in RBCs results in the formation of protrusions from the RBC cell membranes that are known as Heinz bodies.

Heinz bodies are commonly seen in cats

When using NMB, thery will appear as a greenish-blue protrusion on the periphery of the RBC

When using DQ, they will appear as a pale blister or colorless area on the edge of the erythrocye.  A thin rim of the cell membrane may be visible at the protrusion.

Target Cells

This distinctive appearance is the result of an increased membrane surface relative to the cell volume.  Target cells have a central area of pigment surrounded by a clear area and then a dense ring of peripheral cytoplasm.

A few may be seen in a normal blood smear.

They may also be a result of technique errors in making and drying the blood smear.

Increased numbers can be associated with anemias, liver disease, and some inherited disorders.

Basophlic Stippling

appears as very small, variably sized granules within the RBC

These granules are retained RNA aggregates

Can be seen in response to lead poisoning

Hypochromasia

red cells have increased central pallor and decreased staining density of the membrane due to decreased hemoglobin content. 

This is commonly due to insufficient iron or blood loss.

This slide is from a kitten infested with fleas resulting in anemia.

Schistocytes are fragmented erythrocytes usually formed as a result of shearing of the red cell by intravascular trauma.

These are seen in cases of DIC (disseminated intravascular coagulation)  where fibrin within the vascular system mechanically cleave the RBC

Vascular neoplasia, severe burns and some organisms may result in Schistocytes as well.

Acanthocytes

RBCs with irregular projections unevenly distributed on the surface.

Projections are variable in length, diameter and shape.

Occur in patients with altered lipid metabolism and consistently in dogs with hemangiosarcoma

Do no confuse with echinocytes that have numerous, short, evenly place projections that are uniform in shape and size.  These are often artifacts caused by slow drying of blood films or underfilling an EDTA tube.  Non-artifactual findings include poisonous snake bites, renal disease, and lymphosacrcoma in dogs.

Howell-Jolly Body

The RBC at the end of the pointer contains a Howell-Jolly body.

Howell Jolly bodies are basophilic inclusions of young erythrocytes representing nuclear remnants.  They are often seen in most domestic species during a regenerative anemia.

In ruminants, they can be confused with an intracellular parasite, Anaplasma sp...

Rouleaux

Rouleaux formation is the grouping of erythrocytes in stacks or columns and is related to charges on the cell membrane

Marked rouleaux formation may be seen in healthy horses.  In addition, rouleaux may be seen in blood that is held too long before preparing the blood smear or has been refrigerated.  Inflammatory disease can result in increased rouleaux formation.

To distinguish rouleaux and agglutination, mix saline and fresh blood then examine the sample microscopically.  Rouleaux will disperse into individual cells while agglutination will remain in clusters.

Observe the rouleaux formation evident in this field.

Agglutination is the unorganized three-dimensional clustering of red blood cells.  The formation of these grape-like clusters is due to antibodies that coat the erythrocytes resulting in the bridging and clumping of the cells.

Agglutination can be seen both macroscopically and microscopically and just be distinguished from rouleaux formation.

Agglutination may be seen in animals with immune-mediated anemia.

Observe the clumps of RBCs in this field.

Spherocytes

Observe the group of small, dense erythrocytes at the end of the pointer.

These "ball shaped" spherocytes do not have the biconcave shape found in normal canine RBCs

Spherocytes are darkly staining red cells with no central pallor.  They are not easily recognized in species other than dogs.  They appear smaller than normal RBCs due to the loss of the normal biconcave shape.

Spherocytes are significant because they suggest immune-mediated destruction of RBCs, resulting in hemolytic anemia.  They may also be seen after transfusion with mismatched blood.

Although spherocytes have a decreased diameter and appear smaller, their volume is not decreased.

Tears after returning from the long voyage of the Beagle, Charles Darwin started to feel not-so-well dizziness, muscle spasms, fatigue and othe symptoms.  Some speculate that the naturalist may have acquired Chagas Disease while he was traveling in South America as humans are readily infected.

This malady is caused by a single-celled parasite known as Trypanosoma cruzi which is carried by biting insects.

Animals such as opossums, bats, and livestock serve as reservoirs for the parasites

This is a disease seen in domestic animals in Va.

Feline reticulocytes

Reticulocytes are immature erythrocytes that contain organelles that are lost as the cell matures.  These organelles clump into visible granules referred to as reticulum when stained with NMB

Cats, unlike any other species, demonstrate 2 morphologic forms of retics.  The aggregate form contains large clumps of reticulum and is similar to reticulocytes found in other species.  The punctuate form, which is unique to cats, contains 2 - 8 small, singluar, basophilic granules.

Anaplasma marginale

• Intracellular blood parasite
• most prevalent tick borne cattle pathogen
• also affects wild ruminants
• causes a hemolytic anemia
• small coccoid granules on the edge of the erythrocytes
• do not always stain well

Do not confuse with Howell - Jolly bodies

Lymph vs. Nucleated RBCs

Observe the cells in this field that have a dark, basophilic, round nucleus.

Note that the cytoplasm of the lymphocyte is lighter blue compared to the cytoplasm of the nucleated RBC.  The cytoplasm of the nucleated RBC is more similar to the opacity and color of the erythrocytes.

In addition, the nucleated RBCs have a nucleus that is denser and often (though not always) eccentrically located.

All automated cell counters incorrectly include nucleated RBCs in the leukocyte count.

This is one excellent reason for looking at a blood smear in addition to using automated counters.

Mycoplasma haemofelis

a bacterial parasite of feline red blood cells that causes hemolytic anemia (feline infectious anemia)

• this organism can be challenging to find on a blood smear.  the following techniques are helpful in increasing the probability of visualizing this organism in an infected cat.
• use no anticoagulant when obtaining the blood sample.  the organism often detach from the red cells in the presence of anticoagulants
• take multiple samples at different times of the day.  the circulating numbers of the organism fluctuate during the day
• samples taken from a capillary bed (ear margin) may show more organisms due to the difficulty of distorted cells passing through the tiny veins

Cytauxzoon felis

observe the intracellular inclusion found in the erythrocyte - note the "signet ring" or "safety pin" shape of the organism

Cytauxzoon felis is a protozoal parasite that causes a fatal disease in domestic cats.  Bob-cats infected with Cytauxzoon are usually asymptomatic

This is no known effective treatment

Babesia gibsoni

one of the several species of Babesia organisms that cause clinical disease in dogs.  As with Babesia canis, anemia with hemolysis is characteristic finding.  This organism is seen in 50% of pit bulls

• add a drop of EDTA blood to a slide
• add a drop of EDTA blood plus 1 ml saline into a test tube and mix gently, then place the drop on the slide
• put a cover slip on both drops
• observe the plain drop of blood and then observe the drop of blood mixed with saline
• if the the drop mixed with saline looks like individual cells than it is rouleaux, if it still looks like clumps of grapes then its agglutination

to determine if the bone marrow is responding appropriately to anemia (regenerative vs nonregenerative) by counting the number of reticulocytes per 1,000 RBCs.

new methylene blue

under oil, count 1,000 RBCs including reticulocytes.  as you count keep the reticulocytes and RBCs separate

• under oil, count the number of RBCs per 1 representative field.  At this step count ALL rbcs including reticulocytes
• multiply the number of rbcs per field by 10 (if this is less than 1000 cells, you will need to count additional fields to reach a minimum of 1000 rbcs)
• next, count only the reticulocytes in the number of fields you have calculated (a minimum of 10, but may be more)
• adjust your reticulocyte count if you have counted > than 1000 RBCs...(total # of reticulocytes observed) x (1000)/(estimated # of RBCs) = # of reticulocytes per 1000 RBCs
• divide the total number of reticulocytes observed by 10 to get the percentage

measured retic. count x (measured PCV)/(normal PCV) = corrected retic. count

an initial measured retic count needs to be corrected to factor in the degree of anemia.  doing so gives a better estimate of whether or not the circulating population of reticulocytes has increased by the expanded production in the bone marrow

multiply the RBC count by the corrected retic count (be sure to convert the % to a decimal form for the corrected retic count - divide by 100 to get the decimal)

• to an empty test tube, add equal amounts of EDTA blood and NMB (2 - 3 drops of each)
• mix and allow to sit for 10 minutes
• after 10 minutes, mix stained sample and make a blood smear
• observe this slide under oil and do the count

(PCV - drop the %/Total RBC count - drop the scientific notation) x 10 = MCV

• dog:  60 - 77 fl
• cat:  39 - 55 fl

• increased MCV:  macrocytosis (reticulocytosis, FeLV in cats)
• decreased MCV:  microcytosis (iron deficiency, chronic blood loss)

(hemoglobin/PCV - drop %) x 100 = MCHC

hypochromia

no, it would be an artifact like rbc clumping

(# WBC evaluated on differential/100 + # nucleated RBCs) x total leukocyte count = corrected WBC count

• EXAMPLE:
• 8 nucleated RBCs seen, total leukocyte count - 10,500/ul
• (100/100 + 8) x 10,500 = 9,722.22 ~ round to 9,722/ul

PCV/3 = HgB g/dl

(PCV/6) x 10^6 = RBC estimate

• color
• pattern
• shape
• size
• inclusions

anisocytosis

polychromasia

• hypochromia
• hemoglobin

reticulocytes

anemia

Heinz bodies